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Mass Spectrometry (Amide H/D Exchange (HDX) (HDX-MS workflow (types of HDX…
Mass Spectrometry
Amide H/D Exchange (HDX)
unlike covalent labelling which also depends the solvent accessibility (probing territory structure), the H/D exchange is probing the secondary structure
only the amide hydrogen is exchanging at measurable rate; can be quenched by dropping pH to 2.5 (or by dropping the temperature)
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advantages
proteins do not require modification (native conformation),
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exchange rate
high exchange rates: loops, turns and solvent accessible regions
low exchange rate: α-helices, β-sheets (hydrogen bonding network) and core of the protein(poor solvent accessibility)
when analysed by MS
there is an initial shift takes place very rapidly, which would be the based line
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HDX-MS workflow
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types of HDX
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local exchange analysis (digest the protein before ionisation) (residue specific information about the exchange upon ligand binding)
global exchange analysis (ionise the entire protein) (ligand interaction inhibit the H/D exchange, information about the affinity )
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Automated Data analysis
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butterfly plot
decrease in exchange rate due to perturbation indicates the perturbation leads to a more ordered conformation (ligand binding site)
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limitation: doesn't tell why the exchange rate changes upon ligand binding. doesn't tell the kinetics (Kd) (can use surface plasmon resonance to determine the Kd; or using different ligand that does not cause allosteric conformational change) (?)
Application
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influence of mutation: exchange rate is different at the mutation site (or possible allosteric change)
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